Stool samples from 836 cases with diarrhea and acute gastroenteritis from the Pediatric ward, Penang General Hospital, were examined for Cryptosporidium oocysts. A dimethyl sulfoxide modified acid fast technique was used for the identification of the parasites. 36 samples or 4.3% were found to be positive for Cryptosporidium. The prevalence of infection was higher (2.39%) in children with diarrhea and vomiting than in children with acute gastroenteritis alone (0.8%). Stool examination and cultures from the Cryptosporidium positive samples revealed no other parasites, rotavirus or enteropathogenic bacteria. This suggests that Cryptosporidium may be an important agent in the causation of diarrhea in young children. A routine laboratory examination for the detection of Cryptosporidium in the search for causal agents of childhood diarrhea in our environment may, therefore, be significant.
Currently, species-specific information on Entamoeba infections is unavailable in Malaysia and is restricted worldwide due to the re-description of pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. Therefore, this cross-sectional study was conducted to provide the first known documented data on the true prevalence of these three species in western Malaysia using a molecular method. Another aim of this study was to determine the association of potential risk factors associated with each Entamoeba sp. A total of 500 stool samples from three Orang Asli tribes were randomly collected. The overall prevalence of E. histolytica, E. dispar and E. moshkovskii determined by microscopy was 18.6% (93/500). Molecular analysis revealed that while most Entamoeba-positive individuals were infected with E. dispar (13.4%), followed by E. histolytica (3.2%) and E. moshkovskii (1.0%), the present findings show low prevalence rates of mixed infections with E. histolytica and E. dispar (2%), E. dispar and E. moshkovskii (1.2%) and association infections of E. histolytica, E. dispar and E. moshkovskii (0.4%). Logistical regression analysis indicates that the dynamics of the transmission of the three Entamoeba spp. was different. Of six statistically significant variables observed in the univariate analysis, three were retained as significant risk factors for E. histolytica infection in the logistical regression model. These factors were (i) not washing hands after playing with soil or gardening (Odds ratio (OR)=4.7; 95% confidence level (CI)=1.38, 16.14; P=0.013), (ii) indiscriminate defecation in the river or bush (OR=5.7; 95% CI=1.46, 21.95; P=0.012) and (iii) close contact with domestic animals (OR=5.4; 95% CI=1.36, 2.51; P=0.017). However, subjects with family members who were infected with E. histolytica/E. dispar/E. moshkovskii (OR=3.8; 95 CI=2.11, 6.86; P<0.001) and those who consumed raw vegetables (OR=1.8; 95% CI=1.01, 3.23; P=0.047) were more likely to be infected with E. dispar. On the other hand, no associated factor was identified with E. moshkovskii infection. Nevertheless, diarrhoea (P=0.002) and other gastroenteritis symptoms (P<0.001) were only associated with E. histolytica infection. The present study provides new insight into the distribution and risk factors of E. histolytica, E. dispar and E. moshkovskii infections among Orang Asli communities in Malaysia. Identifying the different risk factors of E. histolytica and E. dispar infections will help in the planning specific strategies in the control and prevention of each infection in the communities. Moreover, it emphasises the need for molecular methods to determine the species-specific prevalence of Entamoeba spp.
The flagellate protozoan parasite, Giardia intestinalis, is widely distributed throughout the world with a high prevalence in developing countries in the tropics and subtropics, including Malaysia. Approximately 200 million people are infected with the parasite globally, with 500,000 new cases reported annually. This cross-sectional study was conducted among three tribes of Orang Asli communities in Selangor, Perak and Pahang states of Malaysia. The main objective was to determine the prevalence of and risk factors for giardiasis. Stool samples were collected from 500 individuals aged between 2 and 74 years (males=219, females=281). The samples were examined with formalin-ether sedimentation and trichrome staining techniques. Socioeconomic data were collected through a pre-tested questionnaire. The overall prevalence of giardiasis was 20.0% with the highest prevalence in the Proto-Malays (33.3%) followed by Negritos (20.1%) and Senois (10.4%). The positive cases showed a decrease with increasing age and most of the positive cases were observed in individuals less than 24 years old. Males had significantly higher prevalence than females (χ(2)=5.283, P=0.022). Logistic regression analysis of the overall population studied and the Senoi tribe confirmed that being a child aged less than 15 years, being male, the consumption of raw vegetables and the presence of other family members infected with G. intestinalis were the main risk factors for giardiasis. The presence of other family members infected with G. intestinalis was the only risk factor highlighted in the Proto-Malay and Negrito tribes. Diarrhoea was significantly associated with giardiasis. However, the cause and effect relationship has yet to be determined. Thus, screening family members and treating the infected individuals are the main strategies that should be adopted by the public health authority in combating this infection in Orang Asli communities as well as health education regarding good personal and food hygiene practises.
Entamoeba histolytica infection is the third-greatest parasitic disease responsible for death in the world. Wild rats harbouring E. histolytica can be the possible reservoir hosts for human amoebiasis. There were numerous studies on prevalence of intestinal parasites among wild rats in Malaysia but none has reported E. histolytica. Rats were captured from Sentul and Chow Kit areas, Kuala Lumpur, Malaysia. The preserved stool samples were used for microscopy examination and molecular analysis. Out of 137 samples collected, 12 were positive for E. histolytica / E. dispar / E. moshkovskii microscopically. Two E. histolytica (1.4%), 1 E. dispar (0.7%) and 6 mixed infections of E. histolytica and E. dispar (4.3%) were detected using PCR. This is the first report of molecular detection of E. histolytica/dispar infection among wild rats in Malaysia. This study provides useful information about the potential risks of zoonotic agents and the importance of developing control measures to prevent zoonotic transmission.
Limited data is available on the prevalence of parasitic infections among the island communities in Malaysia with most studies performed between 1960s-1980s. This study was conducted to determine the current prevalence status of parasitic infections among communities living in Pangkor Island Peninsular Malaysia. A total of 131 stool and 298 serum samples were collected and subjected to microscopic examination for intestinal protozoa and helminths and detection of Toxoplasma gondii antibodies using commercial ELISA kits respectively. In addition, thin and thick peripheral blood films were microscopically screened for the presence of Plasmodium spp. and microfilariae respectively. The overall prevalence of intestinal parasitic infections among Pangkor Island community was 9.9% (13/131) with T. trichiura (5.3%) being the most common intestinal parasite detected. Toxoplasmosis was reported in almost 60% of the community with the seroprevalence being significantly high among females (64.7%) compared to males (52.8%) (p=0.038). None of those examined samples were infected with intestinal sarcocystosis, malaria and filariasis. This study revealed that the prevalence of intestinal parasitic infections among Pangkor Island community has been greatly reduced compared to that reported 35 years ago. Massive improvements in the socioeconomic status, personal hygiene, water facilities and sanitation may have contributed to the low prevalence of parasitic infections in this community. Nevertheless, further studies still need to be performed to determine the possible risk factors for the high prevalence of toxoplasmosis in this community.
Intestinal parasitic infections are among the most common diseases affecting mankind causing major public health problems to billions of people living in developing countries. The aim of this study is to determine the prevalence of intestinal parasites in various communities residing in different habitats in Malaysia and compare the findings with 101 studies conducted over the past 42 years (1970-2013). A cross-sectional study design was conducted with the aid of a questionnaire to collect relevant information about the study population. Faecal samples were examined using the direct smear and formal ether sedimentation techniques. A total of 342 children were examined amongst whom 24.6% were positive for intestinal parasitic infections. Results showed that 32.3% of rural children, 20.6% of urban squatters and 5.4% of children from flats were positive for one or more parasites. The most common parasite encountered was Trichuris trichiura (20.2%) followed by Ascaris lumbricoides (10.5%) and hookworm (6.7%). No case of hookworm was reported in urban children whereas 12.2% of rural children were positive. The most common protozoan parasite detected was Entamoeba coli (3.2%) followed by Giardia intestinalis (1.8%), Entamoeba histolytica (1.8%) and Blastocystis hominis (1.2%). Nearly one-fifth (18.4%) of the children had single infection followed by double (12.0%) and triple infections (1.2%). Orang Asli (indigenous) children (44.3%) had the highest infection rate followed by Indians (20.2%), Malays (14.0%) and Chinese (11.9%). Twenty-eight studies carried out on plantation communities with regards to intestinal parasitic infections in Malaysia from 1970 to 2013 showed a steady decline in the prevalence rate ranging from 95.0% in the seventies to 37.0 % in 2012. Intestinal parasitic infections were more common in Orang Asli communities with prevalence ranging from over 90% in the seventies and fluctuating below 70% in most studies between 2000 to 2013 except for two studies that showed a prevalence of 98.2% and 100%. The prevalence rate among urban squatters, urban residents and those living in flats showed dramatic decrease in prevalence rate.
A survey was undertaken to investigate the prevalence of intestinal and blood parasites among wild rats in urban area of Kuala Lumpur, Malaysia. A total of 137 stool and blood samples were collected from wild rats from Sentul and Chow Kit areas. Five species of rats were captured and supplied by Kuala Lumpur City Hall. The most common was Rattus rattus diardii (Malayan Black rat), 67%, followed by Rattus norvegicus (Norway rat), 10%, Rattus argentiventer (rice-field rat), 10%, Rattus tiomanicus (Malaysian field rat), 9% and Rattus exulans (Polynesian rat), 4%. Rattus rattus diardii is commonly known to live in human environment and they are normally identified as pests to human community. More male rats were captured (61%) compared to female (39%). Out of 137 samples, 81.8% samples were positive with intestinal parasites, with 86.2% from Sentul area and 78.5% from Chow Kit area. Six different parasites were detected. The most common intestinal helminth parasite detected was Nippostrongylus brasiliensis (80.3%), followed by Hymenolepis nana (23.4%), Capillaria hepatica (13.9%) and Hymenolepis diminuta (2.9%). Intestinal protozoan detected was Entamoeba histolytica/E. dispar (8.8%). Trypanosoma lewisi (1.5%) was the only blood parasite detected.
We report a case of Hymenolepis diminuta infection in a 2-year-old Malaysian child. This case was initially reported as 'normal' after the examination of proglottids shed from the anus of the child at a private laboratory on two occasions. The putative proglottids shed was then referred to the Parasite Southeast Asia Diagnostic (Para:SEAD) Laboratory, Department of Parasitology, Faculty of Medicine, University of Malaya for further examination. Microscopic examination confirmed that the child was infected with H. diminuta based on the characteristic eggs found in the proglottids. She was treated with a single dose praziquantel (20 mg/kg of body weight) and recovered well.
Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.
Bovine lungworm Dictyocaulus viviparus is highly endemic in temperate regions. However, the occurrence of the lungworm has not been reported in any South East Asian country. The main aim of the present study was to detect the presence of lungworm in cattle in peninsular Malaysia and to examine the morphology of the parasite. A cross-sectional study was carried out in which 602 animals from four large scale government cattle farms and one dairy smallholder farm were sampled. In addition, 283 lungs from 11 abattoirs around the country were examined. Faecal samples were examined using the Baermann technique while post-mortem examination was performed on the lungs. Approximately 5% of faecal samples and 1% of lungs were positive for lungworm. Based on the morphology of adult lungworm, eggs and first stage larvae, Malaysian bovine lungworms were D. viviparus.
Parasites and bacteria are reported in the faeces of birds in the current study. Fresh faecal samples of the large-billed crow (Corvus spp.) were collected from the study site at Bangsar, an urban setting in Kuala Lumpur, Malaysia. These samples were transported to laboratory and analysed for parasites and bacteria. Pre-prepared XLD agar plates were used for culturing the bacteria in the laboratory. Using the API 20ETM Test Strips, 9 different species of bacteria were identified belonging to the family Enterobacteriacea. They were Citrobacter freundii, Enterobacter cloacae, Proteus mirabilis, Klebsiella pneumoniae, Kluyvera ascorbata, Salmonella arizonae, Salmonella typhi, Shigella flexneri and Shigella sonnei. The protozoan parasites detected include Cryptosporidium spp., Cyclospora spp., Blastocystis spp., and Capillaria hepatica and Ascaris lumbricoidus ova. Environmental air samples collected on agar plates using an air sampler in the area only produced fungal colonies. Some of these pathogens found in the crows are of zoonotic importance, especially Cryptosporidium, Blastocystis, Cyclopsora, Salmonella, Shigella and Kluyvera. The finding of Kluyvera spp. in crows in our current study highlights its zoonotic potential in an urban setting.
The Corona pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) calls on the Saudi government to take action to control the infection. The government closed borders, prohibited travel, limited outdoor movements, and told primary and secondary care facilities to reduce all regular non-urgent health services. It is not known whether these measures have impacted the prevalence of parasitic intestinal infections. This study has therefore been carried out to investigate this issue. Dataset of 217 stool samples submitted to the King Faisal Medical Complex (KFMC) Microbiology Laboratory in Taif, Saudi Arabia for parasitological examination during the pandemic (January-June 2020) and 649 samples submitted during the corresponding months of the previous year (January-June 2019) were extracted and analyzed. Overall, 24.1% (209/866) of samples were parasitespositives; 26.6% (173/649) before and 16.5% (36/217) during the pandemic, with 79% reduction. There was a significant difference in gender-parasitism between the two periods where the majority of parasitism were for males (p<0.001). Infections were frequent in patients aged 5- 14 years both before (84/649; 12.9%) and during (12/217; 5.5%) the pandemic, with significant difference observed between the two cohorts (p<0.002). Moreover, the majority of infected patients were non-Saudi (67.9%; 142/209), with a significant difference in nationality reported, (p=0.024). Protozoa were identified in 21.8% (189) of all samples investigated, of which, Blastocystis hominis, Entamoeba coli, Giardia lamblia, Entamoeba histolytica/dispar and Cryptosporidium species were identified in 6.1% (53), 5.4% (47), 5.0% (44), 2.8% (25), and 2.3% (20), respectively. Helminths were diagnosed in 2.3% (20/866) of samples. Eggs of hookworm, Ascaris, Taenia spp, and Hymenolepis nana were detected in 0.9% (8), 0.5% (5), 0.3% (3) and 0.4% (4), respectively. In parallel with our research hypothesis, a substantial decrease in the burden of intestinal parasitic infections was recorded with the lock-down measures taken during the Corona pandemic.
In Soil-Transmitted Helminth (STH) control programs, microscopic examination is applied as a standard method for detecting the presence and the number of STH eggs. The time limitations of fresh specimen processing, especially for an accurate quantitative diagnosis, cause the specimen processing to be delayed or should be performed at a referral laboratory. This deferment requires preservatives to keep the stool integrity without reducing the accuracy. The aims of this study were: 1) to compare the proportion of positive samples and the intensity of A. lumbricoides, T. trichiura, and hookworm infection based on the examination of fresh samples and stool preserved by 10% formalin for >12 months and 2) to determine the most reliably accurate between Kato-Katz and mini-FLOTAC methods in detecting A. lumbricoides, T. trichiura, and hookworm eggs in preserved stools both qualitatively and quantitatively. Seventy-eight (78) stool samples were examined by mini-FLOTAC, and KatoKatz methods. Proportion of positive samples of A. lumbricoides, T. trichiura, and hookworms in fresh and in >=12 months 10% formalin preserved stools had no significant difference. Helminths density (eggs per gram of stool/EPG) in fresh samples was fewer compared to EPG in preserved samples (p <0.05) which leads to a lower proportion of moderate and high level groups in fresh stools samples compared to those in preserved samples (p <0.05). In preserved samples, as qualitative method, mini-FLOTAC detected more A. lumbricoides and T. trichiura eggs than Kato-Katz, while hookworm eggs were detected more by Kato-Katz than the miniFLOTAC. As a quantitative detection, Kato-Katz showed higher calculation of STH EPG than mini-FLOTAC. Using 10% formalin preservation for stool samples, the STH eggs' morphology could still be well identified. Homogenization process and low number of samples tested, were acknowledged as the limitation of this study.
The influx of low skilled migrant workers to Malaysia from low socio-economic countries where gastrointestinal parasitic infections are prevalent has raised concerns about transmission to the local population. Three methods for detection (serology, microscopy and molecular techniques) were utilized to identify Entamoeba infections amongst the targeted cohort and determine risk factors associated with infection. Serological screening of 484 migrant workers from five working sectors in Peninsular Malaysia using IgG4 ELISA based on the rPPDK antigen showed an overall seroprevalence of 7.4% (n = 36; CL95 = 5.3-10.1%) with only one factor statistically associated with seropositivity of anti-amoebic antibodies, i.e. years of residence in Malaysia (χ2 1 = 4.007, p = 0.045). Microscopic examination of 388 faecal samples for protozoan cysts and trophozoites showed a slightly higher prevalence (11.6%; n=45; CL95: 8.4-14.8%). Meanwhile, amplification of the 16S rDNA gene detected two species i.e. Entamoeba dispar (23/388; 5.9%; CL95: 3.6-8.3%) and E. histolytica (11/388; 2.8%; CL95: 1.2-4.5%) and mixed infections with both parasites in only three samples (3/388; 0.8%; CL95: 0.2-2.2%). Entamoeba dispar infection was significantly associated with those employed in food and domestic services (χ2 4 = 12.879, p = 0.012). However, none of the factors affected the prevalence of E. histolytica infection. Despite the low prevalence of E. histolytica in faecal samples of the study cohort, the presence of this pathogenic parasite still poses potential public health risks and calls for tighter control strategies based on better availability of chemotherapeutic treatment and accessibility to appropriate health education.
This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.
Children with severe Trichuris trichiura infection pose significant social, clinical, and therapeutic problems in Malaysia. Thirty such children were investigated, and mebendazole was found to be safe and effective in the treatment of severe trichuriasis but it had to be given for a longer period than currently recommended. A poor correlation was found between egg load and worm burden in these children. Direct visualization of the rectal and colonic mucosa was the most reliable method of assessing severity in untreated cases, and response to treatment. The eggs of Trichuris that had been exposed to mebendazole were morphologically altered and not viable when incubated. This may be of considerable epidemiological importance.